Self-amplifying RNA enables rapid, durable, integration-free programming of hiPSCs.

Area of Science:

• Stem cell biology
• Molecular biology
• Biotechnology

Background:

• Genetic modification of human induced pluripotent stem cells (hiPSCs) is crucial for studying cellular processes in differentiation and disease.
• Traditional genetic engineering methods for hiPSCs are laborious and can lead to issues like karyotypic abnormalities or transgene silencing.
• Self-amplifying RNA (saRNA) presents an integration-free alternative for achieving durable transgene expression.

Purpose of the Study:

• To evaluate saRNA as a method for sustained transgene expression in hiPSCs.
• To demonstrate the utility of saRNA for cell-fate programming and functional reporter expression.
• To assess the application of saRNA in tracking cardiomyocyte maturation and drug responses.

Main Methods:

• Delivery of transcription factors and functional reporters into hiPSCs using saRNA.
• Utilizing saRNA for forward programming of hiPSCs to Ngn2-induced neurons.
• Employing saRNA encoding a jRCaMP1b reporter for differentiation into 3D cardiac spheroids.

Main Results:

• saRNA enabled sustained transgene expression in hiPSCs for several weeks.
• Efficient forward programming to Ngn2-induced neurons was achieved using saRNA.
• Sustained expression of a jRCaMP1b reporter allowed for long-term monitoring of calcium dynamics during cardiomyocyte differentiation.
• The saRNA system facilitated tracking of cardiomyocyte maturation and responses to drugs over weeks.

Conclusions:

• saRNA provides a robust method for sustained transgene expression in hiPSCs without genomic integration.
• This technology supports integration-free cell-fate programming and the measurement of functional reporters in relevant model systems.
• saRNA is a promising tool for advancing research in stem cell biology, disease modeling, and therapeutic development.